1. Technical Field
This invention is in the field of biochemical engineering. More particularly, it concerns a method of oxidizing fully reduced cysteine-containing microbially produced proteins in a controlled manner so that they have disulfide bridging identical to their naturally occurring counterparts.
2. Background Art
When active proteins that contain one or more disulfide bridges are produced microbially via genetic engineering techniques, the synthetic protein is made by the microorganism in a reduced form lacking disulfide bridging or in the form of oligomers that are made in the cell by uncontrolled thiol-disulfide interchange reactions. Tietze, F., Anal Biochem (1969) 27:502. If it is desirable or necessary that the synthetic protein have the same primary structure as its native counterpart, the biochemical engineer is faced not only with the problem of separating the protein from the microorganism culture, but also the problems of reducing oligomers and/or oxidizing the reduced synthetic protein so that it assumes the primary structure of the native protein. Previous oxidations of synthetic microbially produced proteins have been uncontrolled and done deliberately by subjecting the protein to oxidizing conditions or incidentally by placing the proteins in an environment in which it is oxidized. Oxidizing the protein in an uncontrolled manner may: result in the formation of undesirable isomers (incorrect intramolecular bridging) or polymers (intermolecular bridging); overoxidation; complicate the separation of the protein from the culture, or reduce the yield of protein having the desired primary structure. In the case of proteins that are intended for therapeutic use, uncontrolled oxidation through purification, formulation or administration yields a nonhomogeneous material that is contaminated with isomers and/or oligomers that may be inactive or antigenic.
The present invention is directed to a process for oxidizing such microbially produced proteins in a selective, controlled manner using an oxidizing agent, preferably o-iodosobenzoic acid, that oxidizes cysteines selectively, such that the desired disulfide bridging is produced in high yield. In this regard, o-iodosobenzoic acid is a well known sulfhydryl reagent that has been used previously to oxidize vicinal cysteines of native proteins selectively. Hellerman, L., et al., J Amer Chem Soc (1941) 63:2551-2552, Chinard, F. P. and Hellerman, L., Methods Biochem Anal (1954) 1:1, and Vallejos, R. H. and Andreo, C. S., FEBS Letters (1976) 61:95-99. Other oxidizing agents for thiol groups in native proteins are described by Guzman Barron, E. S., Advan Enzymol (1951) 11:223-226 and Teh-Yung Liu, The Proteins (1978) Vol III, 255-263, Academic Press, N.Y. To the best of applicants' knowledge, prior use of o-iodosobenzoic acid and other oxidizing agents as selective oxidants for sulfhydryl groups in proteins has been for analytical purposes. Applicants know of no prior art concerning the use of such oxidants in preparative processes to carry out controlled oxidation of synthetic microbially produced proteins.